2.9. Conjugated Effect of Saccharomycin with Sulfur Dioxide (SO2) on B. bruxellensis Culturability

Simulated wines were prepared using the SGM medium (pH 3.5) mentioned in Section 2.2, modified in its sugars solution to contain just 4.5 g/L of fructose. Ethanol was added to this modified-SGM to obtain simulated wines with 10%, 12%, 13% and 14% (v/v), respectively, with final pH values of 3.5. Each simulated wine was artificially contaminated with 5 × 10³ cells/mL of B. bruxellensis in a final volume of 300 µL. First, the inhibitory effects of ethanol and SO₂ were analyzed in separate, i.e., simulated wines without SO₂ but with 10%, 12%, 13% and 14% (v/v) ethanol, respectively, were used to evaluate the ethanol effect on B. bruxellensis growth; simulated wines without ethanol but with 25, 50, 100 and 150 mg/L of potassium metabisulfite (PMB) (Sigma-Aldrich, Missouri, EUA) (concentrations equivalent to 0.16, 0.33, 0.66 and 1 mg/L of molecular SO₂, at pH 3.5) were used to assess the SO₂ effect on B. bruxellensis growth. Then, the synergistic effect of SO₂ with ethanol was tested using simulated wines at all ethanol levels (i.e., at 10%, 12%, 13% and 14% (v/v) ethanol), each of them supplemented with 25, 50, 100 and 150 mg/L of PMB (Sigma-Aldrich, Missouri, EUA). Finally, the conjugated effect of saccharomycin (obtained as described in Section 2.7) with SO₂ was evaluated on B. bruxellensis growth using the simulated wines at all ethanol levels (i.e., at 10%, 12%, 13% and 14% (v/v) ethanol), each of them supplemented with 0.25, 0.5 and 1 mg/mL of saccharomycin together with PMB at 25 and 50 mg/L, respectively. All growth-assays were performed in triplicates in 96 well-microplates and incubated in a Multiskan-GO spectrophotometer (Thermo-Fisher Scientific Inc., Waltham, MA, USA) at 30 °C, under strong agitation. Cell growth was followed by optical density measurements (at 590 nm) in a Microplate Reader (Dinex Technologies Inc., Chantilly, VA, USA) and by CFU counts. For CFU counts, 10 µL of samples were taken and after appropriate dilution (decimal serial dilution method) 100 µL were plated onto YEPD-agar plates, as described in the Section 2.4.1. Whenever colonies could not be detected in agar-plates inoculated with diluted samples, 100 µL of sample were directly plated onto YEPD-agar plates. Thus, the detection limit of the CFU method for results presented in Section 3.2.2 was 10 CFU/mL.